SummaryDuring erythrocyte invasion, the Plasmodium falciparum Ring-infected erythrocyte surface antigen (RESA) establishes specific interactions with spectrin. Based on analysis of strains with a large chromosome 1 deletion, RESA has been assigned several functions, none of which is firmly established. Analysis of parasites with a disrupted resa1 gene and isogenic parental or resa3 -disrupted controls confirmed the critical role of RESA in the surface reactivity of immune adult sera on glutaraldehyde-fixed ring stages. Absence of RESA did not influence merozoite invasion or erythrocyte membrane rigidity, was associated with a modest increase of cytoadhesion to CD36 under conditions of flow, but resulted in marked susceptibility to heat shock. resa1 -KO-infected erythrocytes were prone to heat-induced vesiculation like uninfected erythrocytes, whereas parental or resa3 -KO infected erythrocytes remained undamaged. Furthermore, a 6 h exposure of ring stages at 41 ∞ ∞ ∞ ∞ C resulted in 33% culture inhibition of resa1 -KO parasites while marginally impacting parental and resa3 -KO parasite growth. This points to a role for RESA in protecting the infected erythrocyte cytoskeleton during febrile episodes. Infection patterns of resa1 -KO and parental parasites in Saimiri sciureus indicated that RESA does not, at least on its own, modulate virulence in the squirrel monkey, as had been previously suggested.

DNA-based immunization of mice by Plasmodium falciparum liver-stage antigen 3 (PfLSA3), a novel highly conserved P. falciparum preerythrocytic antigen, was evaluated. Animals developed a dominant Th1 immune response (high gamma interferon T-cell responses and predominance of immunoglobulin G2a) to each of three recombinant proteins spanning the molecule. We have exploited the immunological cross-reactivity of PfLSA3 with its putative homologue on sporozoites of the rodent parasite Plasmodium yoelii, and we show for the first time that responses induced by PfLSA3 in mice significantly protect against a heterologous challenge by P. yoelii sporozoites. These results support a significant effect of DNA-induced immune responses on preerythrocytic stages.

Three recombinant proteins spanning the Plasmodium falciparum liver-stage Ag-3 (LSA-3) were used to immunize Aotus monkeys. The proteins were delivered subcutaneously without adjuvant, adsorbed onto polystyrene 0.5 ? m particles at a concentration of 2 ? g per immunization. Control animals received glutathione-S-transferase formulated similarly. Animals were challenged as late as 5 months after the last immunization, by intravenous inoculation of 100,000 P. falciparum sporozoites of a strain heterologous to the one from which the immunogens were derived. Sterile protection was achieved in three of the five immunized monkeys but in none of four controls. Antibodies were at low titer, but reacted with the native parasite protein and were boosted by parasite challenge. Ag-specific IFN-+ secretion was detectable in all LSA-3-immunized animals in response to the LSA-3-derived Ag. The protection was apparently associated with high levels of IFN-+ production in response to in vitro recall Ag. These results lend support to the vaccine potential of LSA-3 indicated by previous results obtained in chimpanzees, as well as the value of yet another Ag-delivery system. They also support the value of the Aotus model for the pre-clinical development of preerythrocytic-stage vaccines.

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We synthesized 17 long synthetic peptides (LSP) spanning the whole 200‐kDa Plasmodium falciparum liver stage antigen‐3 (LSA3), an antigen that induces protection in chimpanzee, and analyzed their immunogenicity in BALB/c mice and their antigenicity in individuals living in a hyper‐endemic malaria area. Our findings show that both specific antibodies and T cell proliferation against most LSA3‐LSP develop in malaria‐exposed adults. All individuals studied had detectable antibodies against a minimum of 6 and a maximum of 15 polypeptides. It is noteworthy that antibody prevalence and titers were as high against non‐repeat as repeat regions. Although the extent of T cell reactivity was lower than that observed for B cells, most of the sequences contained at least one T helper epitope, indicating that the majority of LSA3‐LSP contain both B and T cell epitopes within the same sequence. Injection of LSA3‐LSP with SBSA2 adjuvant in mice, showed strong immunogenicity for most of them, eliciting both T cell responses and specific antibody production. While all the peptides were immunogenic for B cells, different patterns of T cell responses were induced. These peptides were thus classified in three sets according to the levels of the T cell proliferative and of the IFN‐γ‐specific responses. Importantly, antibodies and T cells against some of the LSP were able to recognize LSA3 native protein on P. falciparum sporozoites. Additionally, some LSP (44–119, 1026–1095, 1601–1712) also contained epitopes recognized by H‐2d class I‐restricted T cells. These results led to the identification of numerous domains that are highly antigenic and immunogenic within the LSA3 protein, and underline the value of the LSP approach for vaccine development.

Immunity against the pre-erythrocytic stages of malaria is the most promising, as it is strong and fully sterilizing. Yet, the underlying immune effectors against the human Plasmodium falciparum pre-erythrocytic stages remain surprisingly poorly known and have been little explored, which in turn prevents any rational vaccine progress. Evidence that has been gathered in vitro and in vivo, in higher primates and in humans, is reviewed here, emphasizing the significant role of IFN-γ, either as a critical immune mediator or at least as a valuable surrogate marker of protection. One may hope that these results will trigger investigations in volunteers immunized either by optimally irradiated or over-irradiated sporozoites, to quickly delineate better surrogates of protection, which are essential for the development of a successful malaria vaccine.

The Plasmodium falciparum liver-stage antigen 3 (LSA3), a recently identified preerythrocytic antigen, induces protection against malaria in chimpanzees. Using antibodies from individuals with hyperimmunity to malaria affinity purified on recombinant or synthetic polypeptides of LSA3, we identified four non-crossreactive B-cell epitopes in Plasmodium yoelii preerythrocytic stages. On sporozoites the P. yoelii protein detected has a molecular mass similar to that of LSA3. T-cell epitopes cross-reacting with P. yoelii were also demonstrated using peripheral blood lymphocytes from LSA3-immunized chimpanzees. In contrast, no cross-reactive epitopes were found in Plasmodium berghei. LSA3-specific human antibodies exerted up to 100% inhibition of in vitro invasion of P. yoelii sporozoites into mouse hepatocytes. This strong in vitro activity was reproduced in vivo by passive transfer of LSA3 antibodies. These results indicate that the homologous epitopes may be biologically functional and suggest that P. yoelii could be used as a model to assess the antisporozoite activity of anti-LSA3 antibodies.The development of a malaria preerythrocytic vaccine has been greatly influenced by the observation that sterile immunity could be experimentally induced in humans by immunization with Plasmodium falciparum radiation-attenuated sporozoites (9). The critical role of liver-stage trophozoites (reviewed in reference 16) led researchers to initiate a detailed study of the antigens expressed in P. falciparum preerythrocytic stages (22). To date, scientists have identified a series of new molecules expressed at sporozoite and/or liver stage (14). Liver-stage antigen 3 (LSA3), expressed on both the sporozoite surface and in the liver forms, was found to be of particular interest. LSA3 was identified by differential screening of immune responses from protected versus nonprotected volunteers (11). A number of dominant B-and T-cell epitopes to which a high prevalence of responses is detected in individuals exposed to malaria or in LSA3-immunized animals were identified (1, 2). The vaccine potential of this molecule has been recently demonstrated in the chimpanzee model, an animal susceptible and fully receptive to P. falciparum preerythrocytic stages and whose immune system is closest to that of humans. In this model, protection against successive challenges with P. falciparum sporozoites was obtained (11). However, the use of this primate for research purposes is severely hampered by cost and ethical constraints.Several homologues of P. falciparum antigens have been identified, through a variety of immunological and molecular cross-reactivity assays, in other Plasmodium species and particularly in those infecting rodents (5,10,12,13,27,28). The identification of structurally and functionally conserved homologues of P. falciparum proteins in rodent malaria species might help to better understand the role of these molecules in the P. falciparum parasite life cycle. This is particularly true for preerythrocytic stages, where the relative ...

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